PE_PGRS33 Contributes to Mycobacterium tuberculosis Entry in Macrophages through Interaction with TLR2

Abstract

PE_PGRS represent a large family of proteins typical of pathogenic mycobacteria whose members are characterized by an N-terminal PE domain followed by a large Gly-Ala repeat-rich C-terminal domain. Despite the abundance of PE_PGRS-coding genes in the Mycobacterium tuberculosis (Mtb) genome their role and function in the biology and pathogenesis still remains elusive. In this study, we generated and characterized an Mtb H37Rv mutant (MtbΔ33) in which the structural gene of PE_PGRS33, a prototypical member of the protein family, was inactivated. We showed that this mutant entered macrophages with an efficiency up to ten times lower than parental or complemented strains, while its efficiency in infecting pneumocytes remained unaffected. Interestingly, the lack of PE_PGRS33 did not affect the intracellular growth of this mutant in macrophages. Using a series of functional deletion mutants of the PE_PGRS33 gene to complement the MtbΔ33 strain, we demonstrated that the PGRS domain is required to mediate cell entry into macrophages, with the key domain encompassing position 140–260 amino acids of PE_PGRS33. PE_PGRS33-mediated entry into macrophages was abolished in TLR2-deficient mice, as well as following treatment with wortmannin or an antibody against the complement receptor 3 (CR3), indicating that PE_PGRS33-mediated entry of Mtb in macrophages occurs through interaction with TLR2.

Development of Potent Inhibitors of the Mycobacterium tuberculosis Virulence Factor Zmp1 and Evaluation of Their Effect on Mycobacterial Survival inside Macrophages

“…Adhered macrophages were infected with M. tuberculosis H37Rv strain at a MOI of 1:1. As described for J774 cells, h MDMs were infected and intracellular mycobacterial CFUs were determined at 4 h and 72 h post‐infection . Compound 1 c was added 24 h post‐infection at different concentrations (20, 6.5, 2.5 μg mL −1 ) while we added a second‐line anti‐TB drug, capreomycin (4 μg mL −1 ) as positive control.…”

Impact of pe_pgrs33 Gene Polymorphisms on Mycobacterium tuberculosis Infection and Pathogenesis

“…Each pe_pgrs33 allele under the control of its native promoter was cloned into the integrative plasmid pMV306 in-frame with the HA epitope sequence (Palucci et al, 2016). The previously generated Mtb Δ33 mutant strain (Palucci et al, 2016) was then complemented with all constructs, according to procedures already described (Cascioferro et al, 2011; Palucci et al, 2016).…”

“…Based on previous evidences which implicated PE_PGRS33 in the pathogenesis of Mtb (Brennan et al, 2001; Palucci et al, 2016) and supported its role in mediating Mtb entry into macrophages and triggering of inflammatory responses in a TLR2-dependent mechanism (Brennan et al, 2001; Basu et al, 2007; Zumbo et al, 2013), here we assessed whether and how natural genetic variations may affect the functionality of PE_PGRS33 in in vitro and in vivo models of TB.…”

Virulence Factors and Pathogenicity of Mycobacterium

“…PE-PGRS and PE-PPE interacts with the TLR-2 on DC and macrophages, inducing: cytokine secretion, necrosis and apoptosis and enhance mycobacterial survival [60]. PE-PGRS33 interaction with TLR mediates macrophage entry [61]. PE-PGRS30 mutant showed an attenuated phenotype, specifically inhibits phagosome-lysosome fusion and showed decreased lung colonization and reduced tissue damage [62].…”